The overall objective of this proposal is to develop a platform that broadly enables the generation of a new class of anti-viral therapeutics that recruit and activate the innate immunity system to attack specifically human cells infected with targeted viral pathogens. The platform deploys a recently discovered transportable diversity generating system to create massive libraries of binding domains built on a scaffold that is naturally a component of MICA, an important ligand for the NKG2D receptor on NK and T-cells of the innate immunity system. The proposed targeting component of the MICA molecule is the (3 domain, which is an immunoglobin-like structure without any known function in MICA other than spacing the NKG2D ligand domain away from the cell surface to which MICA is normally attached. Thus, the (3 domain is an ideal candidate for diversification by the proprietary diversity generator to create a massive library of targeting domains. The isolation of the desired (3 domain of MICA involves expressing the diversified library of (3 as a fusion protein on the surfaces of E. coli and selecting the bacteria that bind to fluorescent nanolipoprotein particles decorated with the intended viral glycoprotein target. The gene encoding the (3 domain with the desired binding properties can then be directly obtained from the isolated E. coli. The system should have very broad applications in the creation of "passive therapeutic vaccines" for acute treatment of viral infections. We have chosen to pursue initially the well characterized lymphocytic choriomeningitis virus (LCMV) and yellow fever virus (YFV), prototypic viruses representing the highly virulent arenavirus and flavivirus pathogens, respectively. Because of their remarkable potency as direct recruiters of NKG2D-bearing innate immunity effector cells, MICA-based specific targeting agents might eventually replace ADCC-dependent therapeutic monoclonal antibodies, including those targeting tumor antigens on cancer cells. PUBLIC HEALTH RELEVANCE: The best protection from a viral infection is a prior infection by the same viral strain;the second best is active vaccination against the infecting strain. Both of these protective mechanisms depend upon the "adaptive" arm of the immunity system, such as antibodies. Adaptive immunity takes many weeks to develop in response to the initial exposure or vaccination. The innate immunity system, on the other hand, is a promptly acting, potent although rather non-specific arm of the immunity system that can be protective against some viruses and bacteria. This proposal involves engineering a molecule called MICA. MICA molecules frequently appear attached to the outside surface of infected cells and there recruit cells of the innate immunity arm to destroy the infected cells before the virus or bacteria can replicate within the infected cells and spread. The goal of the project is to convert innate immunity into a system that more closely resembles the adaptive immunity in its specificity and potency but yet can act promptly after being administered medically. MICA serves as a "key" (ligand) to the "lock" (receptor) on Natural Killer (NK) cells that once engaged will kill the MICA decorated diseased cell. However, many virulent viruses have found means of preventing the MICA decoration of their host cells so as to thwart the innate immunity and protect their host cells long enough for them, the viruses, to replicate and spread. This project proposes to convert natural human MICA into soluble molecules that after intravenous administration can find and bind specifically to infected cells and thereby recruit the cells of innate immunity such as NK cells to attack. The ability to create a massive library of diverse soluble MICA molecules with different binding specificities will enable the creation of a platform with broad applications for the acute treatment of specific infectious diseases.